The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the Caucasian population. It affects approximately 1 in 2,500 live births in North America (Boat et al, The Metabolic Basis of Inherited Disease, 6th ed, pp 2649-2680, McGraw Hill, NY (1989)). Approximately 1 in 25 persons are carriers of the disease. The major symptoms of cystic fibrosis include chronic pulmonary disease, pancreatic exocrine insufficiency, and elevated sweat electrolyte levels. The symptoms are consistent with cystic fibrosis being an exocrine disorder. Although recent advances have been made in the analysis of ion transport across the apical membrane of the epithelium of CF patient cells, it is not clear that the abnormal regulation of chloride channels represents the primary defect in the disease.
The gene for CF has been localized to a 250,000 base pair genomic sequence present on the long arm of chromosome 7. This sequence encodes a membrane-associated protein called the “cystic fibrosis transmembrane regulator” (or “CFTR”). There are greater than 1000 different mutations in the CFTR gene, having varying frequencies of occurrence in the population, presently reported to the Cystic Fibrosis Genetic Analysis Consortium. These mutations exist in both the coding regions (e.g., ΔF508, a mutation found on about 70% of CF alleles, represents a deletion of a phenylalanine at residue 508) and the non-coding regions (e.g., the 5T, 7T, and 9T mutations correspond to a sequence of 5, 7, or 9 thymidine bases located at the splice branch/acceptor site of intron 8) of the CFTR gene. Comparison of the CFTR genomic and cDNA sequences confirms the presence of 27 exons. The exons are numbered 1-27 as shown in NCBI Reference Sequence accession no. NM_000492.3. Each intron is flanked by the consensus GT-AG splice-site sequence as previously reported (Zielenski, et al., (1991) Genomics 10, 214-228).
Methods for detecting CFTR gene mutations have been described. See e.g., Audrezet et al., “Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms” Hum Mutat. 2004 April; 23(4):343-57; PCT WO 1004/040013 A1 and corresponding US application #20040110138; titled “Method for the detection of multiple genetic targets” by Spiegelman and Lem; US patent application No. 20030235834; titled “Approaches to identify cystic fibrosis” by Dunlop et al.; and US patent application No. 20040126760 titled “Novel compositions and methods for carrying out multiple PCR reactions on a single sample” by N. Broude.
Currently, however, multiple different analysis and/or detection methods must be employed in order to accurately obtain comprehensive sequence data. For example, traditional Sanger sequencing methodology may be employed to determine the presence or absence of mutations involving a small number of nucleotides in the CFTR gene. Sanger sequencing, though, is unable to detect large deletions and duplications such as those involving one or more exons. As a result, additional methods such as quantitative fluorescent polymerase chain reaction (QF-PCR) are needed to detect these larger types of mutations.
Accordingly, improved methods are needed to efficiently detect the variety of CFTR gene defects which underlie CF and to simultaneously capture both dosage data (e.g., gene copy number) and sequence data. Moreover, improved methods are needed for detecting rare mutations in the CFTR gene. Ideally, methods that can detect multiple classes of CFTR mutations such as those involving small base changes (e.g., missense mutations, nonsense mutations, small insertions or deletions and/or splice-site mutations) and those involving larger deletions and/or duplications in a single assay are desirable.